A Novel Strategy for Introducing Exogenous Bcl-2 into Neuronal Cells: The Cre/loxP System-Mediated Activation of Bcl-2 for Preventing Programmed Cell Death Using Recombinant Adenoviruses

doi:10.1006/mcne.1998.0703

Molecular and Cellular Neuroscience

Volume 12, Issues 1–2, September 1998, Pages 65-78

https://doi.org/10.1006/mcne.1998.0703 Get rights and content

Abstract

We have established a novel strategy for introducing exogenous Bcl-2 into neuronal cells that is mediated by Cre/loxP recombination using recombinant adenoviral vectors. An on/off-switching cassette for Bcl-2 (CALNLbcl-2) was designed to express Bcl-2 by recombinase Cre-mediated excisional deletion of a spacer DNA flanked by a pair ofloxP sites. Exogenous Bcl-2 was clearly induced in PC12 cell lines carrying CALNLbcl-2 after infection with recombinant adenovirus producing recombinase Cre (AxCANCre). Dual infection with both AxCANCre and a recombinant adenovirus bearing CALNLbcl-2 showed efficient delivery of exogenous Bcl-2 into a hybrid motoneuronal cell line and primary chicken spinal motoneurons. The delivery of foreign Bcl-2 promoted survival of motoneurons in medium either containing or lacking trophic support. Thus, this strategy for delivery of exogenous Bcl-2 will be useful for studying neuronal death as well as for introducing foreign genes into postmitotic neurons under the control of recombinase Cre.

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To study the Bcl-2 expression mediated by the Cre/loxP recombination system and its effect on prevention of apoptosis in olfactory epithelium. Adenoviral vectors with cassette for Bcl-2 (AxCALNLBcl-2) and Cre recombinase (AxCANCre) were applied to mouse olfactory epithelium by intranasal instillation. The effect of exogenous Bcl-2 expression on prevention of apoptosis of olfactory receptor neurons was investigated using an apoptosis model induced by bulbectomy. The Bcl-2 product was expressed not only in the olfactory receptor neurons but also in the supporting cells. Although statistical analysis did not show significant difference, the number of apoptotic cells in the infected olfactory epithelium on post-bulbectomy day 2 was lower than that of control and the number of survived mature olfactory receptor neurons in the infected olfactory epithelium on post-bulbectomy day 5 was higher than that of control. Although further studies are required for clinical application, the results of our study suggest that this strategy may be able to deliver exogenous Bcl-2 for the treatment of degeneration of olfactory receptor neurons.
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Citation Excerpt :
Constitutively active MEK (da-MEK) was subcloned into pAxCAwt (39), an expression cosmid cassette that was created from the human type 5 adenovirus genome from which the E1A, E1B, and E3 regions were deleted and replaced with an expression unit under the control of the CAG promoter (40). The myr-Akt fragment was subcloned into pAxCALNLw, a Cre-inducible expression cassette (41). Akt-AA (Akt T308A/S473A), which contains a hemagglutinin tag at its N terminus and works as a dominant-negative mutant of Akt (42), was kindly provided by Dr. M. Kasuga (Kobe University).
Tumor necrosis factor-α is thought to be one of the most important inflammatory cytokines associated with the demyelinating disease multiple sclerosis. We determined whether neurotrophins could protect oligodendrocytes from tumor necrosis factor-α-mediated cytotoxicity. Among the neurotrophins tested, nerve growth factor was most effective at preventing cell death. Nerve growth factor also prevented the tumor necrosis factor-induced loss of mitochondrial membrane potential. Overexpression of constitutively active Akt, a downstream target of phosphatidylinositol 3-kinase, but not of constitutively active MEK, protected oligodendrocytes from tumor necrosis factor-induced injury. Moreover, overexpression of dominant-negative Akt negated the protective effects of nerve growth factor on tumor necrosis factor-mediated oligodendrocyte cytotoxicity. These findings indicate that the Akt pathway is crucial in nerve growth factor-mediated oligodendrocyte protection.
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Neonatal rats were exposed to ethanol throughout gestation, or during the early postnatal period (postnatal days 4–10 (P4–10)), and enzyme-linked immunoabsorbent assays were subsequently conducted in order to assess nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) protein content in hippocampus, septum, cortex/striatum and cerebellum. These determinations revealed that following prenatal ethanol treatment, there were significant ethanol-induced increases in NGF in P1 cortex/striatum, but no changes in any of the three neurotrophic factors (NTFs) in the other brain regions. Cortex/striatal NGF protein returned to control levels by P10. Following early postnatal exposure, BDNF was elevated in hippocampus and cortex/striatum (assessed on P10), and NGF was also enhanced in cortex/striatum at this age. Hippocampal and cortex/striatal BDNF returned to control levels by P21, but cortex/striatal NGF levels remained enhanced at this age. This NTF did not differ in ethanol and control animals by P60, however. The possible significance of elevated levels of NTFs as a function of ethanol exposure is discussed, and it is speculated that while such alterations could play a protective role, increases in these substances during critical developmental periods could also prove to be deleterious, and could even contribute to certain of the neuropathologies which have been observed following developmental ethanol exposure.
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Bcl-2 overexpression prevents neuronal death after injury or neurotrophic factor-deprivation but the biochemical consequences of survival maintenance by Bcl-2 have hardly been explored. We show that unlike NGF, adenovirally delivered hBcl-2 supports the survival of over 80% of the neurons without activating ERK and Akt phosphorylation, or suppressing JNK phosphorylation, or enhancing cell growth. However, the proapoptotic protein BAD, whose phosphorylation is induced by NGF, is degraded in NGF-deprived neurons expressing hBcl-2, while the level of Bcl-xL remains unaffected. Interestingly, degradation of BAD protein is prevented by the pan-caspase inhibitor Boc.Asp(OMe)fmk. We propose that NGF-deprivation promotes dephosphorylation of BAD while hBcl-2 facilitates its release into the cytoplasm where it is degraded by noncaspase, Boc.Asp(O-Me)fmk-inhibitable proteases. The potential importance of BAD degradation is suggested by our finding that overexpressed BAD kills NGF-maintained sympathetic neurons by apoptosis, while hBcl-2 prevents BAD-induced death.
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Regular ArticleA Novel Strategy for Introducing Exogenous Bcl-2 into Neuronal Cells: The Cre/loxP System-Mediated Activation of Bcl-2 for Preventing Programmed Cell Death Using Recombinant Adenoviruses

Abstract

Cell

Adv. Cell Neurobiol

Mol. Brain Res.

Gene

Trends Genet.

J. Biol. Chem.

Neuron

Neuron

Gene

Curr. Opin. Neurobiol.

Site-specific recombination mediated by an adenovirus vector expressing the Cre recombinase protein: A molecular switch for control of gene expression

J. Virol.

Aurintricarboxylic acid rescues PC12 cells and sympathetic neurons from cell death caused by nerve growth factor deprivation: Correlation with suppression of endonuclease activity

J. Cell Biol.

Bcl-2 affects survival but not neuronal differentiation of PC12 cells

J. Neurosci.

Targeted transduction of CNS neurons with adenoviral vectors carrying neurotrophic factor genes confers neuroprotection that exceeds the transduction population

J. Neurosci

Intrastriatal injection of an adenoviral vector expressing glial-cell-line-derived neurotrophic factor prevents dopaminergic neuron degeneration and behavioral impairment in a rat model of Parkinson disease

Proc. Natl. Acad. Sci. USA

Protection of retinal ganglion cells from natural and axotomy-induced cell death in neonatal transgenic mice overexpressing bcl-2

J. Neurosci.

Bcl-2 promotes regeneration of served axons in mammalian CNS

Nature

bcl-2 overexpression reduces apoptotic photoreceptor cell death in three different retinal degenerations

Proc. Natl. Acad. Sci. USA

Dopaminergic neurons protected from degeneration by GDNF gene therapy

Science

Neonatal motoneurons overexpressing thebcl

Proc. Natl. Acad. Sci. USA.

Early stages of motor neuron differentiation revealed by expression of homeobox geneIslet

Science

bcl

Proc. Natl. Acad. Sci. USA

Prevention of programmed cell death of sympathetic neurons by thebcl

Science

Prevention of motoneuron death by adenovirus-mediated neurotrophic factors

J. Neurosci. Res

Regular Article
A Novel Strategy for Introducing Exogenous Bcl-2 into Neuronal Cells: The Cre/loxP System-Mediated Activation of Bcl-2 for Preventing Programmed Cell Death Using Recombinant Adenoviruses